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1. A Thermostable Monoacylglycerol Lipase from Marine Geobacillus sp. 12AMOR1: Biochemical Characterization and Mutagenesis Study.

Author

Tang W, Lan D, Zhao Z, Li S, Li X, and Wang Y

Subjects
Amino Acid Sequence, Catalysis, Cloning, Molecular methods, Diglycerides genetics, Escherichia coli genetics, Esterification genetics, Hydrogen-Ion Concentration, Hydrolysis, Recombinant Proteins genetics, Sequence Alignment, Substrate Specificity genetics, Triglycerides genetics, Geobacillus genetics, Monoacylglycerol Lipases genetics, Mutagenesis genetics
Abstract

Lipases with unique substrate specificity are highly desired in biotechnological applications. In this study, a putative marine Geobacillus sp. monoacylglycerol lipase (GMGL) encoded gene was identified by a genomic mining strategy. The gene was expressed in Escherichia coli as a His-tag fusion protein and purified by affinity chromatography with a yield of 264 mg per liter fermentation broth. The recombinant GMGL shows the highest hydrolysis activity at 60 °C and pH 8.0, and the half-life was 60 min at 70 °C. The GMGL is active on monoacylglycerol (MAG) substrate but not diacylglycerol (DAG) or triacylglycerol (TAG), and produces MAG as the single product in the esterification reaction. Modeling structure analysis showed that the catalytic triad is formed by Ser97, Asp196 and His226, and the flexible cap region is constituted by residues from Ala120 to Thr160. A mutagenesis study on Leu142, Ile145 and Ile170 located in the substrate binding tunnel revealed that these residues were related with its substrate specificity. The k cat /K m value toward the pNP-C6 substrate in mutants Leu142Ala, Ile145Ala and Ile170Phe increased to 2.3-, 1.4- and 2.2-fold as compared to that of the wild type, respectively.

Published
2019
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2. Description of compensatory gyrA mutations restoring fluoroquinolone susceptibility in Mycobacterium tuberculosis

Author

Stéphanie Petrella, Alexandra Aubry, Nicolas Veziris, Hélène Ferrand, Claudine Mayer, Aurélie Bouige, Alix Pantel, Stéphanie Matrat, Wladimir Sougakoff, Université Pierre et Marie Curie - Paris 6 (UPMC), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Parts of this paper were presented at the ‘International Conference on Antimicrobial Agents and Chemotherapy’, USA, 2011 (C1-626), and at the EMBO Conference ‘Tuberculosis 2012’, France, 2012 (TUBERCULOSIS2012/000269)., Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre National de Référence des Mycobactéries et de la Résistance aux Antituberculeux [CHU Pitié-Salpêtrière], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, Bactériologie-Hygiène [CHU Pitié-Salpêtrière] ], and CHU Pitié-Salpêtrière [APHP]

Subjects
0301 basic medicine, MESH: Mycobacterium tuberculosis, DNA Mutational Analysis, Mutant, Antitubercular Agents, medicine.disease_cause, DNA gyrase, MESH: Recombinant Proteins, Suppression, Genetic, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Pharmacology (medical), MESH: DNA Mutational Analysis, MESH: Suppression, Genetic, MESH: Inhibitory Concentration 50, Mutation, MESH: Microbial Sensitivity Tests, biology, Chemistry, MESH: Escherichia coli, Recombinant Proteins, 3. Good health, MESH: Mutagenesis, Site-Directed, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, DNA Gyrase, DNA supercoil, Fluoroquinolones, medicine.drug, MESH: DNA Gyrase, Microbiology (medical), 030106 microbiology, Mutation, Missense, Microbial Sensitivity Tests, Microbiology, Mycobacterium tuberculosis, Inhibitory Concentration 50, 03 medical and health sciences, Drug Resistance, Bacterial, MESH: Drug Resistance, Bacterial, Escherichia coli, medicine, Humans, Pharmacology, MESH: Mutation, Missense, MESH: Humans, Mutagenesis, MESH: Fluoroquinolones, biology.organism_classification, MESH: Antitubercular Agents, Mutagenesis, Site-Directed, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, Ofloxacin
Abstract

International audience; OBJECTIVES:Resistance to fluoroquinolones (FQs) in Mycobacterium tuberculosis (Mtb) is mainly due to mutations in DNA gyrase (GyrA2B2), with the most common substitutions located at positions 90 and 94 in GyrA. Two clinical MDR Mtb (MDR-TB) strains harbouring an A90E or D94N substitution in GyrA were found to be surprisingly susceptible to FQs (ofloxacin MIC ≤2 mg/L). We studied the impact of the additional GyrA substitutions found in these strains (T80A and T80A + A90G, respectively) on FQ susceptibility.METHODS:Mutants of interest were generated by site-specific mutagenesis of GyrA alleles. WT and mutant TB DNA gyrase subunits were overexpressed in Escherichia coli and purified, and the in vitro susceptibility to FQs of their DNA supercoiling reaction was studied.RESULTS:IC50s of mutant gyrase complexes bearing GyrA D94N and A90E were 3- to 36-fold higher than WT IC50s, whereas IC50s of gyrase bearing T80A + A90G + D94N and T80A + A90E were close to the WT IC50s.CONCLUSIONS:We demonstrated that substitutions T80A and A90G restore FQ susceptibility when associated with a substitution implicated in high-level FQ resistance. Line probe assay misclassification of MDR-TB strains as pre-XDR or XDR can be corrected by sequence analysis of gyrA.

Published
2016

3. β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

Author

Jörg Vogel, Ivan Matic, S. Crussard, Jesús Blázquez, Luisa Laureti, Zeynep Baharoglu, Didier Mazel, F. Darfeuille, Alexandro Rodríguez-Rojas, Arnaud Gutierrez, Heni Abida, Robustesse et évolvabilité de la vie (U1001), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología [Madrid] (CNB-CSIC), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC)-Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Plasticité du Génome Bactérien - Bacterial Genome Plasticity (PGB), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Régulations Naturelles et Artificielles (ARNA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Bordeaux Ségalen [Bordeaux 2], Julius-Maximilians-Universität Würzburg (JMU), This work was supported by the FP7-HEALTH-F3-2010-241476 and ANR-09-BLAN-0251 grants. A.G. was supported by a fellowship from the French Ministry of Science and Education. D.M. was supported by the Institut Pasteur, Centre National de la Recherche Scientifique (CNRS-UMR 3525), the French National Research Agency (ANR-08-MIE-016), LABEX IBEID and the European Union Seventh Framework Programme EvoTAR. Z.B. was supported by a postdoctoral fellowship from the DIM Malinf. J.B. was supported by grants PI10/00105 (FIS) and REIPI (RD06/0008), both from Ministerio de Ciencia e Innovacion, Instituto de Salud Carlos III., We thank Dan Andersson’s lab (Uppsala University, Sweden) for providing the anti-DinB antibody, Marie Francoise Norel (Pasteur Institute, Paris) for providing the anti-RpoS antibody and Paul Modrich’s lab for providing the anti-MutS antibody., I.M., A.G., D.M., J.B., F.D. and J.V. designed the research and wrote the paper. A.G., L.L., H.A. and S.C. performed experiments with E. coli. Z.B. performed experiments and analysed data with V. cholerae. A.R.R. performed experiments and analysed data with P. aeruginosa. F.D. and J.V. performed in vitro experiments with SdsR and analysed data., ANR-09-BLAN-0251,bactadapt(2009), ANR-08-MIEN-0016,IIRE,Integrase d'integron recombinaison et expression(2008), European Project: 241476,EC:FP7:HEALTH,FP7-HEALTH-2009-single-stage,PAR(2010), European Project: 282004,EC:FP7:HEALTH,FP7-HEALTH-2011-single-stage,EVOTAR(2011), HOFFMANN, Isabelle, Blanc - - bactadapt2009 - ANR-09-BLAN-0251 - Blanc - VALID, Maladies Infectieuses et environnement - Integrase d'integron recombinaison et expression - - IIRE2008 - ANR-08-MIEN-0016 - MIE - VALID, Predicting antibiotic resistance - PAR - - EC:FP7:HEALTH2010-04-01 - 2013-03-31 - 241476 - VALID, Evolution and Transfer of Antibiotic Resistance - EVOTAR - - EC:FP7:HEALTH2011-10-01 - 2015-09-30 - 282004 - VALID, Centro Nacional de Biotecnologia, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Julius-Maximilians-Universität Würzburg [Wurtzbourg, Allemagne] (JMU)

Subjects
DNA Replication, medicine.drug_class, [SDV]Life Sciences [q-bio], Antibiotics, General Physics and Astronomy, Sigma Factor, MESH: DNA Replication, Biology, beta-Lactams, medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Microbiology, 03 medical and health sciences, Antibiotic resistance, Bacterial Proteins, MESH: beta-Lactams, MESH: Anti-Bacterial Agents, Escherichia coli, medicine, Vibrio cholerae, MESH: Bacterial Proteins, 030304 developmental biology, MESH: Mutagenesis, 0303 health sciences, Multidisciplinary, Bacteria, 030306 microbiology, Pseudomonas aeruginosa, MESH: Escherichia coli, Mutagenesis, MESH: Reactive Oxygen Species, MESH: Sigma Factor, General Chemistry, biochemical phenomena, metabolism, and nutrition, Anti-Bacterial Agents, 3. Good health, [SDV] Life Sciences [q-bio], MESH: Bacteria, Regulon, MESH: Pseudomonas aeruginosa, bacteria, Reactive Oxygen Species, rpoS, MESH: Vibrio cholerae
Abstract

Antimicrobials: mismatch excels when ampicillin runs low. [Nat Rev Microbiol. 2013]; International audience; Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.

Published
2013